Publications by authors named "Andre J van Wijnen"

498 Publications

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Bone 2021 Oct 23;154:116234. Epub 2021 Oct 23.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.
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http://dx.doi.org/10.1016/j.bone.2021.116234DOI Listing
October 2021

Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing.

JBMR Plus 2021 Oct 23;5(10):e10520. Epub 2021 Jun 23.

Department of Orthopedic Surgery Mayo Clinic Rochester MN USA.

Bromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow-derived mesenchymal stem cells via a conditional Brd4 allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector-based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad-BMP2). Importantly, Brd4 depletion in MC3T3-E1 and human adipose-derived mesenchymal stem cells (AMSCs) transduced with Ad-BMP2 enhances osteogenic differentiation of naïve MC3T3-E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector-based gene therapies. © 2021 The Authors. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8520065PMC
October 2021

Hinfp is a guardian of the somatic genome by repressing transposable elements.

Proc Natl Acad Sci U S A 2021 Oct;118(41)

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. We show here that loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of in this process is , which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging.
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http://dx.doi.org/10.1073/pnas.2100839118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8521681PMC
October 2021

Are Serum Ion Levels Elevated in Pediatric Patients With Metal Implants?

J Pediatr Orthop 2021 Oct 7. Epub 2021 Oct 7.

Departments of Orthopedic Surgery Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN Department of Orthopedics, National Clinical Research Center for Orthopedics, Sports Medicine & Rehabilitation, General Hospital of Chinese PLA Department of Geriatric Medicine, Peking University People's Hospital, Beijing, China Sanford Orthopedics and Sports Medicine, Sioux Fall, SD.

Background: Previous studies report elevated serum titanium (Ti) levels in children with spinal implants. To provide additional data on this topic, we sought to assess serum ion levels at multiple timepoints in pediatric patients with growing spine devices, spinal fusion instrumentation, and extremity implants placed for fracture treatment. We hypothesized that serum Ti, cobalt (Co), and chromium (Cr) levels would be elevated in pediatric patients with growing spine devices compared with patients with extremity implants.

Methods: Pediatric patients undergoing any primary spine implant placement, those with spine implant revision or removal surgery and patients with other appendicular implant removal had serum Ti, Co, and Cr ion levels drawn at the time of surgery. Fifty-one patients (12 growing spine devices, 13 fusions, and 26 extremity implants) had one set of labs, 31 of whom had labs drawn both preoperatively and postoperatively. Biopsies obtained from tissue specimens at the time of implant revision were analyzed histologically for the presence of metal debris and macrophage activity.

Results: Patients with growing spine implants had elevated serum Ti (3.3 vs. 1.9 ng/mL, P=0.01) and Cr levels (1.2 vs. 0.27 ng/mL, P=0.01) in comparison to patients with fusion rods or extremity implants. With respect to patients with extremity implants, patients with growing spine devices had elevated serum Ti (3.3 vs. 0.98 ng/mL, P=0.013), Co (0.63 vs. 0.26 ng/mL, P=0.017), and Cr levels (1.18 vs. 0.26 ng/mL, P=0.005). On matched pairs analysis, patients who had labs drawn before and after spine implantation had significant increase in serum Ti levels (0.57 vs. 3.3 ng/mL, P=0.02). Histology of tissue biopsies adjacent to growing spine implants showed presence of metal debris and increased macrophage activity compared with patients with extremity implants.

Conclusion: Serum Ti, Co, and Cr levels are elevated in children with spinal implants compared with those with extremity implants, particularly in those with growing spine devices. However, the clinical significance of these findings remains to be determined.

Level Of Evidence: Level II-prospective comparative study.
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http://dx.doi.org/10.1097/BPO.0000000000001957DOI Listing
October 2021

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Gene 2020 Dec 16;763S:100027. Epub 2020 Jan 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.
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http://dx.doi.org/10.1016/j.gene.2020.100027DOI Listing
December 2020

Differentially Expressed Functional LncRNAs in Human Subjects With Metabolic Syndrome Reflect a Competing Endogenous RNA Network in Circulating Extracellular Vesicles.

Front Mol Biosci 2021 17;8:667056. Epub 2021 Aug 17.

Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, United States.

Metabolic syndrome (MetS), a collective cluster of disease risk factors that include dyslipidemia, obesity, inflammation, hypertension, and insulin resistance, affects numerous people worldwide. Accumulating studies have shown that long non-coding RNAs (lncRNAs) serve as competing endogenous RNAs (ceRNAs) to play essential roles in regulating gene expression in various diseases. To explore the role of lncRNAs as ceRNAs in MetS, we examined a MetS-associated network in circulating extracellular vesicles (EVs) collected from the systemic blood of MetS and control patients ( = 5 each). In total, 191 differentially expressed lncRNAs, 1,389 mRNAs, and 138 miRNAs were selected for further analysis. Biological processes and pathway functional enrichment analysis were performed based on the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The lncRNA/mRNA/miRNA ceRNA network was constructed by Cytoscape v3.8 based on the DE-RNAs and included 13 lncRNAs, 8 miRNAs, and 64 mRNAs. MetS patients showed elevated body weight, glucose, blood pressure, insulin, liver injury, and inflammatory marker levels. We found that lncRNAs reflect a ceRNA network that may regulate central cellular processes and complications of MetS, including cancer. These findings suggest that MetS alters the interactions among the ceRNA network components in circulating EVs and that this cargo of circulating EVs may have potential translational ramifications for MetS.
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http://dx.doi.org/10.3389/fmolb.2021.667056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415968PMC
August 2021

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

PLoS One 2021 22;16(7):e0254426. Epub 2021 Jul 22.

Musculoskeletal Research Center, Washington University School of Medicine, St. Louis, MO, United States of America.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0254426PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297882PMC
November 2021

The dynamic broad epigenetic (H3K4me3, H3K27ac) domain as a mark of essential genes.

Clin Epigenetics 2021 07 8;13(1):138. Epub 2021 Jul 8.

CancerCare Manitoba Research Institute, CancerCare Manitoba, Winnipeg, MB, R3E 0V9, Canada.

Transcriptionally active chromatin is marked by tri-methylation of histone H3 at lysine 4 (H3K4me3) located after first exons and around transcription start sites. This epigenetic mark is typically restricted to narrow regions at the 5`end of the gene body, though a small subset of genes have a broad H3K4me3 domain which extensively covers the coding region. Although most studies focus on the H3K4me3 mark, the broad H3K4me3 domain is associated with a plethora of histone modifications (e.g., H3 acetylated at K27) and is therein termed broad epigenetic domain. Genes marked with the broad epigenetic domain are involved in cell identity and essential cell functions and have clinical potential as biomarkers for patient stratification. Reducing expression of genes with the broad epigenetic domain may increase the metastatic potential of cancer cells. Enhancers and super-enhancers interact with the broad epigenetic domain marked genes forming a hub of interactions involving nucleosome-depleted regions. Together, the regulatory elements coalesce with transcription factors, chromatin modifying/remodeling enzymes, coactivators, and the Mediator and/or Integrator complex into a transcription factory which may be analogous to a liquid-liquid phase-separated condensate. The broad epigenetic domain has a dynamic chromatin structure which supports frequent transcription bursts. In this review, we present the current knowledge of broad epigenetic domains.
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http://dx.doi.org/10.1186/s13148-021-01126-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8264473PMC
July 2021

Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis.

Int J Mol Sci 2021 Jun 19;22(12). Epub 2021 Jun 19.

Department of Mechanical & Biomedical Engineering, Boise State University, 1910 University Drive, MS-2085, Boise, ID 83725, USA.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.
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http://dx.doi.org/10.3390/ijms22126580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234021PMC
June 2021

Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Biochem Biophys Res Commun 2021 08 16;567:72-78. Epub 2021 Jun 16.

Department of Oral Biology, Rutgers School of Dental Medicine, NJ, USA. Electronic address:

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.
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http://dx.doi.org/10.1016/j.bbrc.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290883PMC
August 2021

The Micro-RNA Cargo of Extracellular Vesicles Released by Human Adipose Tissue-Derived Mesenchymal Stem Cells Is Modified by Obesity.

Front Cell Dev Biol 2021 20;9:660851. Epub 2021 May 20.

Department of Medicine, Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, United States.

Obesity is a chronic disease that interferes with normal repair processes, including adipose mesenchymal stem/stromal cells (ASCs) function. ASCs produce extracellular vesicles (EVs) that activate a repair program in recipient cells partly via their micro-RNA (miRNA) cargo. We hypothesized that obesity alters the miRNA expression profile of human ASC-derived EVs, limiting their capacity to repair injured cells. Human ASCs were harvested from obese and age- and gender-matched non-obese (lean) subjects during bariatric or cosmetic surgeries, respectively ( = 5 each), and their EVs isolated. Following high-throughput sequencing analysis, differentially expressed miRNAs were identified and their gene targets classified based on cellular component, molecular function, and biological process. The capacity of human lean- and obese-EVs to modulate inflammation, apoptosis, as well as mitogen-activated protein kinase (MAPK) and Wnt signaling in injured human proximal tubular epithelial (HK2) cells was evaluated . The number of EVs released from lean- and obese-ASCs was similar, but obese-EVs were smaller compared to lean-EVs. Differential expression analysis revealed 8 miRNAs upregulated (fold change > 1.4, < 0.05) and 75 downregulated (fold change < 0.7, < 0.05) in obese-EVs vs. lean-EVs. miRNAs upregulated in obese-EVs participate in regulation of NFk-B and MAPK signaling, cytoskeleton organization, and apoptosis, whereas those downregulated in obese-EVs are implicated in cell cycle, angiogenesis, and Wnt and MAPK signaling. Treatment of injured HK2 cells with obese-EVs failed to decrease inflammation, and they decreased apoptosis and MAPK signaling significantly less effectively than their lean counterparts. Obesity alters the size and miRNA cargo of human ASC-derived EVs, as well as their ability to modulate important injury pathways in recipient cells. These observations may guide development of novel strategies to improve healing and repair in obese individuals.
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http://dx.doi.org/10.3389/fcell.2021.660851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173369PMC
May 2021

Surface Roughness of Titanium Orthopedic Implants Alters the Biological Phenotype of Human Mesenchymal Stromal Cells.

Tissue Eng Part A 2021 Aug 16. Epub 2021 Aug 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

Metal orthopedic implants are largely biocompatible and generally achieve long-term structural fixation. However, some orthopedic implants may loosen over time even in the absence of infection. fixation failure is multifactorial, but the fundamental biological defect is cellular dysfunction at the host-implant interface. Strategies to reduce the risk of short- and long-term loosening include surface modifications, implant metal alloy type, and adjuvant substances such as polymethylmethacrylate cement. Surface modifications (e.g., increased surface rugosity) can increase osseointegration and biological ingrowth of orthopedic implants. However, the localized responses of cells to implant surface modifications need to be better characterized. As an model for investigating cellular responses to metallic orthopedic implants, we cultured mesenchymal stromal/stem cells on clinical-grade titanium disks (Ti6Al4V) that differed in surface roughness as high (porous structured), medium (grit blasted), and low (bead blasted). Topological characterization of clinically relevant titanium (Ti) materials combined with differential mRNA expression analyses (RNA-seq and real-time quantitative polymerase chain reaction) revealed alterations to the biological phenotype of cells cultured on titanium structures that favor early extracellular matrix production and observable responses to oxidative stress and heavy metal stress. These results provide a descriptive model for the interpretation of cellular responses at the interface between native host tissues and three-dimensionally printed modular orthopedic implants, and will guide future studies aimed at increasing the long-term retention of such materials after total joint arthroplasty.
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http://dx.doi.org/10.1089/ten.TEA.2020.0369DOI Listing
August 2021

Human meniscus allograft augmentation by allogeneic mesenchymal stromal/stem cell injections.

J Orthop Res 2021 May 10. Epub 2021 May 10.

Orthopedics and Sports Medicine, Mayo Clinic, Rochester, Minnesota, USA.

Meniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof-of-concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh-frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock-injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100-µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc-like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle-shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.
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http://dx.doi.org/10.1002/jor.25074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8578587PMC
May 2021

Multiple pharmacological inhibitors targeting the epigenetic suppressor enhancer of zeste homolog 2 (Ezh2) accelerate osteoblast differentiation.

Bone 2021 09 30;150:115993. Epub 2021 Apr 30.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Center for Regenerative Medicine, Mayo Clinic, Rochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.
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http://dx.doi.org/10.1016/j.bone.2021.115993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217219PMC
September 2021

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

Gene 2021 Jul 20;788:145662. Epub 2021 Apr 20.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States. Electronic address:

Introduction: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis.

Methods: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy.

Results: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6.

Conclusions: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.
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http://dx.doi.org/10.1016/j.gene.2021.145662DOI Listing
July 2021

Acquired Idiopathic Stiffness After Contemporary Total Knee Arthroplasty: Incidence, Risk Factors, and Results Over 25 Years.

J Arthroplasty 2021 08 1;36(8):2980-2985. Epub 2021 Apr 1.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN.

Background: Acquired idiopathic stiffness (AIS) remains a common failure mode of contemporary total knee arthroplasties (TKAs). The present study investigated the incidence of AIS and manipulation under anesthesia (MUA) at a single institution over time, determined outcomes of MUAs, and identified risk factors associated with AIS and MUA.

Methods: We identified 9771 patients (12,735 knees) who underwent primary TKAs with cemented, modular metal-backed, posterior-stabilized implants from 2000 to 2016 using our institutional total joint registry. Mean age was 68 years, 57% were female, and mean body mass index was 33 kg/m. Demographic, surgical, and comorbidity data were investigated via univariate Cox proportional hazard models and fit to an adjusted multivariate model to access risk for AIS and MUA. Mean follow-up was 7 years.

Results: During the study period, 456 knees (3.6%) developed AIS and 336 knees (2.6%) underwent MUA. Range of motion (ROM) increased a mean of 34° after the MUA; however, ROM for patients treated with MUA was inferior to patients without AIS at final follow-up (102° vs 116°, P < .0001). Significant risk factors included younger age (HR 2.3, P < .001), increased tourniquet time (HR 1.01, P < .001), general anesthesia (HR 1.3, P = .007), and diabetes (HR 1.5, P = .001).

Conclusion: Acquired idiopathic stiffness has continued to have an important adverse impact on the outcomes of a subset of patients undergoing primary TKAs. When utilized, MUA improved mean ROM by 34°, but patients treated with MUA still had decreased ROM compared to patients without AIS. Importantly, we identified several significant risk factors associated with AIS and subsequent MUA.

Level Of Evidence: Level III, retrospective comparative study.
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http://dx.doi.org/10.1016/j.arth.2021.03.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292170PMC
August 2021

Biomechanical, histological, and molecular characterization of a new posttraumatic model of arthrofibrosis in rats.

J Orthop Res 2021 Apr 19. Epub 2021 Apr 19.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

Experimental analyses of posttraumatic knee arthrofibrosis utilize a rabbit model as a gold standard. However, a rodent model of arthrofibrosis offers many advantages including reduced cost and comparison with other models of organ fibrosis. This study aimed to characterize the biomechanical, histological, and molecular features of a novel posttraumatic model of arthrofibrosis in rats. Forty eight rats were divided into two equal groups. An immobilization procedure was performed on the right hind limbs of experimental rats. One group was immobilized for 4 weeks and the other for 8 weeks. Both groups were remobilized for 4 weeks. Limbs were studied biomechanically via assessment of torque versus degree of extension, histologically via whole knee specimen, and molecularly via gene expression of posterior capsular tissues. Significant differences were observed between experimental and control limbs at 4 N-cm of torque in the 4-week (knee extension: 115° ± 8° vs. 169° ± 17°, respectively; p = 0.007) and 8-week immobilization groups (knee extension: 99° ± 12° vs. 174° ± 9°, respectively; p = 0.008). Histologically, in each group experimental limbs demonstrated increased posterior capsular thickness and total area of tissue when compared to control limbs (p < 0.05). Gene expression values evaluated in each group were comparable. This study presents a novel rat model of arthrofibrosis with severe and persistent knee contractures demonstrated biomechanically and histologically. Statement of clinical significance: Arthrofibrosis is a common complication following contemporary total knee arthroplasties. The proposed model is reproducible, cost-effective, and can be employed for translational investigations studying the pathogenesis of arthrofibrosis and efficacy of neoadjuvant pharmacologic agents.
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http://dx.doi.org/10.1002/jor.25054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8523596PMC
April 2021

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

Diabetes 2021 07 15;70(7):1561-1574. Epub 2021 Apr 15.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.
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http://dx.doi.org/10.2337/db19-1268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336004PMC
July 2021

Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis.

Stem Cell Res Ther 2021 04 14;12(1):240. Epub 2021 Apr 14.

Division of Nephrology and Hypertension, Mayo Clinic, 200, First Street SW, Rochester, MN, 55902, USA.

Background: Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes.

Methods: Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O) or hypoxia (1%O) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-β-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues.

Results: Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-β-gal activity decreased in both animal groups.

Conclusions: These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.
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http://dx.doi.org/10.1186/s13287-021-02310-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048283PMC
April 2021

Elevated Expression of Plasminogen Activator Inhibitor (PAI-1/SERPINE1) is Independent from rs1799889 Genotypes in Arthrofibrosis.

Meta Gene 2021 Jun 5;28. Epub 2021 Mar 5.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.

Arthrofibrosis is characterized by excessive extracellular matrix deposition in patients with total knee arthroplasties (TKAs) and causes undesirable joint stiffness. The pathogenesis of arthrofibrosis remains elusive and currently there are no diagnostic biomarkers for the pathological formation of this connective tissue. Fibrotic soft tissues are known to have elevated levels of plasminogen activator inhibitor-1 (PAI-1) (encoded by ), a secreted serine protease inhibitor that moderates extracellular matrix remodeling and tissue homeostasis. The 4G/5G insertion/deletion (rs1799889) is a well-known polymorphism that directly modulates PAI-1 levels. Homozygous 4G/4G allele carriers typically have higher PAI-1 levels and may predispose patients to soft tissue fibrosis (e.g., liver, lung, and kidney). Here, we examined the genetic contribution of the rs1799889 polymorphism to musculoskeletal fibrosis in arthrofibrotic (n = 100) and non-arthrofibrotic (n = 100) patients using Sanger Sequencing. Statistical analyses revealed that the allele frequencies of the rs1799889 polymorphism are similar in arthrofibrotic and non-arthrofibrotic patient cohorts. Because the fibrosis related rs1799889 polymorphism is independent of arthrofibrosis susceptibility in TKA patients, the possibility arises that fibrosis of joint connective tissues may involve unique genetic determinants distinct from those linked to classical soft tissue fibrosis.
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http://dx.doi.org/10.1016/j.mgene.2021.100877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011541PMC
June 2021

Gut-microbiota modulation: The impact of thegut-microbiotaon osteoarthritis.

Gene 2021 Jun 27;785:145619. Epub 2021 Mar 27.

Departments of Bioengineering, the University of Illinois at Chicago, Chicago, IL, USA; Jesse Brown Veterans Affairs Medical Center (JBVAMC) at Chicago, IL, USA. Electronic address:

Osteoarthritis (OA) is one of the most common medical conditions affecting > 300 million people globally which represents the formidable public health challenge. Despite its clinical and financial ramifications, there are currently no approved disease modifying OA drugs available and symptom palliation is the only alternative. Currently, the amount of data on the human intestinal microbiome is growing at a high rate, both in health and in various pathological conditions. With an increase in the amount of the accumulated data, there is an expanded understanding that the microbiome provides compelling evidence of a link between thegut microbiomeand development ofOA. The microbiota management tools of probiotics and/or prebiotics or symbiotic have been developed and indeed, commercialized over the past few decades with the expressed purpose of altering the microbiota within the gastrointestinal tract which could be a potentially novel intervention to tackle or prevent OA. However, the mechanisms how intestinal microbiota affects the OA pathogenesis are still not clear and further research targeting specific gut microbiota or its metabolites is still needed to advance OA treatment strategies from symptomatic management to individualized interventions of OA pathogenesis. This article provides an overview of the various preclinical and clinical studies using probiotics and prebiotics as plausible therapeutic options that can restore the gastrointestinal microbiota and its impact on the OA pathogenesis. May be in the near future the targeted alterations of gut microbiota may pave the way for developing new interventions to prevent and treat OA.
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http://dx.doi.org/10.1016/j.gene.2021.145619DOI Listing
June 2021

Low-Dose Tamoxifen Induces Significant Bone Formation in Mice.

JBMR Plus 2021 Mar 20;5(3):e10450. Epub 2021 Jan 20.

Department of Medicine University of North Carolina Chapel Hill NC USA.

Use of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β-catenin in 28-day-old male and female Prrx1-CreER/β-catenin mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β-catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild-type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose-dependent manner analyzed by micro-computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%,  < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%,  < 0.0005) and 58% ± 3.8% (269%,  < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: (224%,  < 0.0001), (166%,  < 0.0001), (223%,  < 0.0001), and (228%,  < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990151PMC
March 2021

Is Essential for Patterning of Multiple Musculoskeletal Tissues but Dispensable for Tendon Differentiation.

Stem Cells Dev 2021 Jun 27;30(11):601-609. Epub 2021 Apr 27.

Research Division, Shriners Hospital for Children, Portland, Oregon, USA.

An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of signaling.
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http://dx.doi.org/10.1089/scd.2020.0209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165461PMC
June 2021

Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial.

Stem Cell Res Ther 2021 03 18;12(1):187. Epub 2021 Mar 18.

Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

Background: Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases.

Methods: In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS).

Results: Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs.

Conclusions: Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.
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http://dx.doi.org/10.1186/s13287-021-02241-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977179PMC
March 2021

Identification of novel therapeutic targets for contrast induced acute kidney injury (CI-AKI): alpha blockers as a therapeutic strategy for CI-AKI.

Transl Res 2021 09 9;235:32-47. Epub 2021 Mar 9.

Vascular and Interventional Radiology Translational Laboratory, Department of Radiology, Mayo Clinic, Rochester, Minnesota; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota; Department of Pulmonary and Critical Care Medicine Mayo Clinic, Rochester, Minnesota. Electronic address:

Iodinated contrast is used for imaging and invasive procedures and it can cause contrast induced acute kidney injury (CI-AKI), which is the third leading hospital-acquired health problem. The purpose of the present study was to determine the effect of α-adrenergic receptor-1b (Adra1b) inhibition by using terazosin on change in kidney function, gene, and protein expression in C57BL/6J male mice, 6-8 weeks with chronic kidney disease (CKD). CKD was induced by surgical nephrectomy. Twenty eight days later, 100-µL of iodinated contrast (CI group) or saline (S group) was given via the carotid artery. Whole-transcriptome RNA-sequencing (RNA-Seq) analysis of the kidneys was performed at day 2. Mice received either 50-µL of saline ip or terazosin (2 mg/kg) in 50-µL of saline ip 1 hour before contrast administration which was continued every 12 hours until the animals were euthanized 2 and 7 days later. The kidneys were removed for gene expression, immunohistochemical analysis, and blood serum analyzed for kidney function. Differential gene expression analysis identified 21 upregulated and 436 downregulated genes (fold change >2; P < 0.05) that were common to all sample (n = 3 for both contrast and saline). We identified Adra1b using bioinformatic analysis. Mice treated with terazosin had a significant decrease in serum creatinine, urinary Kim-1 levels, HIF-1α, apoptosis, and downstream Adrab1 genes including Ece1, Edn1, pMAPK14 with increased cell proliferation. Contrast exposure upregulated Adra1b gene expression in HK-2 cells. Inhibition of Adra1b with terazosin abrogated Ece1, Edn1, and contrast-induced Fsp-1, Mmp-2, Mmp-9 expression, and caspase-3/7 activity in HK-2 cells.
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http://dx.doi.org/10.1016/j.trsl.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328880PMC
September 2021

Absence of signature inflammatory markers in synovial fluid for total knee arthroplasties revised for arthrofibrosis.

Orthop Traumatol Surg Res 2021 05 24;107(3):102870. Epub 2021 Feb 24.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, United States. Electronic address:

Introduction: Current diagnostic criteria for arthrofibrosis are limited. Since many patients will be aspirated during their clinical course, synovial fluid analysis may supplement current diagnostic criteria for arthrofibrosis. The goal of this study was to determine a unique synovial fluid and inflammatory marker profile for diagnosing arthrofibrosis.

Hypothesis: Patients with arthrofibrosis following total knee arthroplasty will have a unique synovial fluid aspirate profile compared to control patients to aid in diagnosis.

Materials And Methods: Between 2013 and 2017, 32 patients (32 total knee arthroplasties [TKAs]) underwent revision TKAs for arthrofibrosis. Fourteen patients had pre-revision aspirations. They were 2:1 matched based on age, sex, body mass index (BMI), and year of revision to 28 patients who underwent aseptic revision TKAs for reasons other than arthrofibrosis (control group). Mean age at revision was 66years, with 64% males.

Results: In TKAs revised for arthrofibrosis, mean total cell count was 828cells/uL. These aspirates contained a mean distribution of 46% macrophages (range: 4-76%), 31% lymphocytes (range: 11-68%), 21% neutrophils (range: 0-75%), 1% other cells (mainly synovial cells; range: 0-11%), and 1% eosinophils (range: 0-7%). There was no significant difference in mean total cell count (p=0.8) or mean distribution of macrophages (p=0.6), lymphocytes (p=0.1), neutrophils (p=0.2), eosinophils (p>0.9), or serum inflammatory markers (p>0.7) when compared to controls. All aspirations were culture negative for infection.

Discussion: The profile of arthrofibrotic synovial fluid aspirates and serum inflammatory marker values were similar to patients revised for non-arthrofibrotic aseptic etiologies. This suggests synovial fluid and serum inflammatory markers in non-infected knees with arthrofibrosis should expect to have characteristics similar to synovial fluid and inflammatory marker profiles in other aseptic diagnoses.

Level Of Evidence: III; retrospective case-control study.
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http://dx.doi.org/10.1016/j.otsr.2021.102870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087631PMC
May 2021

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Stem Cells Dev 2021 03;30(6):325-336

Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24 cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24 and CD24 BMSCs identified a limited number of genes with increased expression in CD24 cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24 cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.
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http://dx.doi.org/10.1089/scd.2021.0027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984936PMC
March 2021

Polysaccharide from Angelica sinensis attenuates SNP-induced apoptosis in osteoarthritis chondrocytes by inducing autophagy via the ERK1/2 pathway.

Arthritis Res Ther 2021 01 30;23(1):47. Epub 2021 Jan 30.

Department of Orthopedics, The Affiliated Changzhou No.2 People's Hospital of Nanjing Medical University, 29 Xinglong Alley, Changzhou, 213003, China.

Objective: Chondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism.

Method: Human primary chondrocytes isolated from the articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3) II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localization, and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile, activation of the ERK 1/2 pathway was determined by western blot. The autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ), and a specific inhibitor of ERK1/2, SCH772984, were used to confirm the autophagic effect of ASP.

Results: The results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocyte proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of the ERK1/2 pathway.

Conclusion: ASP markedly rescued SNP-induced apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP as a potential therapeutic supplementation for OA treatment.
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http://dx.doi.org/10.1186/s13075-020-02409-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847159PMC
January 2021

Gene expression profiles of human adipose-derived mesenchymal stem cells dynamically seeded on clinically available processed nerve allografts and collagen nerve guides.

Neural Regen Res 2021 Aug;16(8):1613-1621

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.

It was hypothesized that mesenchymal stem cells (MSCs) could provide necessary trophic factors when seeded onto the surfaces of commonly used nerve graft substitutes. We aimed to determine the gene expression of MSCs when influenced by Avance® Nerve Grafts or NeuraGen® Nerve Guides. Human adipose-derived MSCs were cultured and dynamically seeded onto 30 Avance® Nerve Grafts and 30 NeuraGen® Nerve Guides for 12 hours. At six time points after seeding, quantitative polymerase chain reaction analyses were performed for five samples per group. Neurotrophic [nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), pleiotrophin (PTN), growth associated protein 43 (GAP43) and brain-derived neurotrophic factor (BDNF)], myelination [peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ)], angiogenic [platelet endothelial cell adhesion molecule 1 (PECAM1/CD31) and vascular endothelial cell growth factor alpha (VEGFA)], extracellular matrix (ECM) [collagen type alpha I (COL1A1), collagen type alpha III (COL3A1), Fibulin 1 (FBLN1) and laminin subunit beta 2 (LAMB2)] and cell surface marker cluster of differentiation 96 (CD96) gene expression was quantified. Unseeded Avance® Nerve Grafts and NeuraGen® Nerve Guides were used to evaluate the baseline gene expression, and unseeded MSCs provided the baseline gene expression of MSCs. The interaction of MSCs with the Avance® Nerve Grafts led to a short-term upregulation of neurotrophic (NGF, GDNF and BDNF), myelination (PMP22 and MPZ) and angiogenic genes (CD31 and VEGFA) and a long-term upregulation of BDNF, VEGFA and COL1A1. The interaction between MSCs and the NeuraGen® Nerve Guide led to short term upregulation of neurotrophic (NGF, GDNF and BDNF) myelination (PMP22 and MPZ), angiogenic (CD31 and VEGFA), ECM (COL1A1) and cell surface (CD96) genes and long-term upregulation of neurotrophic (GDNF and BDNF), angiogenic (CD31 and VEGFA), ECM genes (COL1A1, COL3A1, and FBLN1) and cell surface (CD96) genes. Analysis demonstrated MSCs seeded onto NeuraGen® Nerve Guides expressed significantly higher levels of neurotrophic (PTN), angiogenic (VEGFA) and ECM (COL3A1, FBLN1) genes in the long term period compared to MSCs seeded onto Avance® Nerve Grafts. Overall, the interaction between human MSCs and both nerve graft substitutes resulted in a significant upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the NeuraGen® Nerve Guide was more pronounced, particularly in the long term period (> 14 days after seeding). These results suggest that MSC-seeding has potential to be applied in a clinical setting, which needs to be confirmed in future in vitro and in vivo research.
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http://dx.doi.org/10.4103/1673-5374.303031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8323683PMC
August 2021
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